SAG (Sensitive to Apoptosis Gene), also known as RBX2/ROC2, is a stress-responsive RING component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase, required for its activity. Our strong preliminary data as well as published results showed that 1) Sag is a gene essential for growth during mouse embryonic development, and Sag-null MEFs or ES cells are growth retarded when cultured in vitro or grown in nude mice, respectively;2) SAG transgenic expression accelerates the growth of DMBA/TPA-induced skin papilloma by inhibiting apoptosis;3) SAG is overexpressed in multiple human cancers, particularly lung cancer, and 4) SAG siRNA silencing suppresses cancer cell growth via inducing apoptosis. However, whether Sag is required for immortalization, transformation, or carcinogenesis, thus serving as a valid cancer target for chemoprevention and therapy, has not been examined. The objectives of this application are to use tissue specific Sag knockout mouse models at physiological settings to study the role of Sag in immortalization and transformation of primary cultures and in carcinogenesis of lung and skin tissues. The central hypothesis is that Sag, upon overexpression, promotes cell growth and inhibits apoptosis via promoting the degradation of tumor suppressive and apoptosis-inducing substrates such as IkB, Nf1, p27, and Noxa, leading to activation of the NFkB and Ras pathways. On the other hand, Sag, upon inactivation by genetic deletion or treatment with small molecule inhibitor MLN4924, causes an accumulation of these substrates to inactivate the NFkB and Ras pathways, resulting in suppression of proliferation and carcinogenesis. Three specific aims are proposed to elucidate the role of Sag in 1) immortalization and transformation of mouse embryonic fibroblasts by E1A/Hras;2) lung carcinogenesis induced by KrasG12D, and 3) skin carcinogenesis induced by DMBA/TPA. IMPACT: our work is highly innovative and of significant impact with translational value by validating SAG E3 ubiquitin ligase as an attractive target for cancer chemoprevention and therapy, thus providing some proof-of-concept evidence for future development of MLN4924, a newly discovered small molecule inhibitor of SCF E3 ligase, as a novel class of chemoprevention and anticancer agents. PUBLIC HEALTH RELEVANCE: Targeted cancer therapy relies on thorough validation of cancer targets. We found that Sag E3 ubiquitin ligase is overexpressed and activated in human cancer, particularly lung cancer. In this study, we will mechanistically elucidate the role of Sag in neoplastic transformation and in carcinogenesis of lung (induced by Kras) and skin (induced by chemical carcinogens). We will test MLN4924, a small molecule inhibitor of Sag E3 ligase against lung cancer at the early and later stages. Thus, this study is of highly translational value by validating Sag E3 ubiquitin ligase and its inhibitor MLN4924 as an attractive chemoprevention and anticancer target and drug, respectively.